Screening kit and method for diagnosing chronic immune dysfunction syndrome

ABSTRACT

The subject invention permits diagnosis of chronic fatigue syndrome (CFS) by analyzing peripheral blood mononuclear cell subset populations and activation markers. A positive diagnosis for CFS is associated with an increase in the percentage of CD8+ cells showing CD38 or HLA-DR cell markers or a decrease in CD11b markers in CD8+ cells. The analysis is preferably performed using fluorochrome-labelled monoclonal antibodies specific for a determinant of the above subset cells and activation markers.

CROSS-REFERENCES

This application is a divisional application of earlier filed U.S.patent application Ser. No. 07/787,389, filed Nov. 4, 1991 issued U.S.Pat. No. 5,426,028, which is a continuation application of earlier filedapplication Ser. No. 07/725,309, filed Jul. 5, 1991 (now abandoned) towhich applications we claim priority under 35 USC §120 and whichapplications are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The invention concerns methods for diagnosing chronic fatigue syndromeas well as cell cultures containing an infectious agent associated withchronic fatigue syndrome.

BACKGROUND

Chronic fatigue syndrome (CFS) is being reported with increasingfrequency in many sections of the United States as well as other partsof the world, including England and Australia. In many patients, CFSbegins with an acute "flu-like" illness and is characterized by adebilitating fatigue lasting for more than 3-6 months; chronic andrecurrent low-grade fever, pharyngitis, adenopathy, myalgia, arthralgia,sleep disorders and mood disorders. A common feature is multilevel braindisorder, reflected in mental changes such as loss of memory, vertigo,and disorientation, often described as "spaciness." Direct measurementsof brain function using, for example, magnetic resonance imaging (MRI)have indicated abnormalities in the central nervous system. Dementia andsigns of "mental clouding" have also been documented throughpsychoneurologic testing.

The infectious agent(s) for CFS is unknown; the agent is suspected to beviral, at least in part because many viral infections are characterizedby chronic fatigue. However, post viral fatigue generally does notpersist for more than a few weeks, which is contrary to the clinicalpicture in CFS. Conclusive evidence of an etiologic association of aparticular known virus to CFS has not been presented, although highlevels of antibodies to Epstein Barr virus (EBV), human herpes virus-6(HHV-6) and the p24 core antigen of HTLV have been reported. Further,certain immunologic abnormalities described in CFS are often found inviral infections, including activation of CD8+ cells. Other immunologicabnormalities observed include decreased function of NK cells, reducedmitogenic responses of lymphocytes and B-cell subset changes.

It is of interest to determine whether there are virologic andimmunologic parameters which are at least substantially specific for CFSso that they can be used as a diagnostic aid in those patients whopresent with symptoms including chronic fatigue. Further, definitivediagnosis of CFS is hampered by the lack of a screening test such as animmune profile or a serologic test to identify the etiologic agent.Identification of the etiologic agent to produce recombinant proteinsthat are safe for use in vaccines, diagnostics, and/or screening of theblood supply is greatly desired.

Relevant Literature

The Centers for Disease Control (CDC) have attempted to define CFS inrecent cases using major and minor criteria. Holmes et al., Ann. Intern.Med. (1988) 108:387-89. CFS has been reported to be associated withEpstein-Barr virus. See, Strauss et al. Ann. Intern. Med. (1985)102:7-16; Buchwald et al. JAMA (1987) 257:2303-7; and Tobi et al.,Lancet (1982) 1:61-4. High antibody levels to HHV-6 observed in some CFSpatients also do not correlate with any particular symptoms nor withvirus isolation. See, for example, Levy et al., The Lancet (1990)335:1047-50; Krueger et al., J. Virol. Methods (1988) 21:125; andWakefield et al., The Lancet (1988) 1:1059. The presence of enteroviralantigens in muscles of people with CFS has been reported, and Coxsackievirus infections are reported to be associated with fatigue. Archard etal., J. Royal Soc. Med. (1988) 81:326-9; and Yousef et al., Lancet(1988) 1:146-50. However, an association of Coxsackie virus with CFS hasnot been confirmed. Miller, BMJ (1991) 302:140. Likewise, the linking ofan HTLV-like virus with CFS, while suggested, has not been confirmed.Palca, Science (1990) 249:1240-41; DeFrietas et al., PNAS (May, 1991).

Decreased function of NK cells, reduced mitogenic response oflymphocytes, B-cell subset changes, and activation of CD8+ cells withCFS have been reported. Kibler et al., J. Clin. Immunol. (1985) 5:46-54;Tosato et al., J. Immunol. (1985) 134:3082-88; Murdoch Nv. Ned. J.(1988) 101:511-2; Caligiuri et al., J. Immunol. (1987) 139:3306-13;Lloyd et al., Med. J. Australia (1989) 151:122-24; Jin et al, Med. J.Aust. (1989) 151:117-19; and Klimas et al., J. Clin. Micro. (1990)28:1403-10. Infected animals and patients suffering or recovering from avariety of acute viral infections frequently display transient immuneabnormalities and chronic fatigue. Notkins et al., Ann. Rev. Microbiol.(1970) 24:525; Rouse and Horchov, Rev. Insect. Dis. (1986) 8:850-73.

SUMMARY OF THE INVENTION

In accordance with the subject invention, methods are provided fordiagnosing chronic fatigue syndrome in a host presenting symptoms whichinclude chronic fatigue. The method involves the step of screeningperipheral blood mononuclear cells (PMC) from a host for changes in PMCmarkers as compared to PMC from a healthy control. Also provided arecell culture procedures for finding the infectious agent associated withCFS. The cell cultures can be used as a source of genomic material forpreparing polynucleotide probes for diagnosis of CFS, as well asantigens and vaccines for therapeutic and diagnostic applications.Propagation of the infectious agent in vitro can be used to identifycell surface antigens associated with the infectious agent and as asource of such antigens.

DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows the percent expression (mean±standard error) of CD11b,CD38 and HLA-DR on CD8+ cells from normals, severely afflicted (group1), recovering (group 2) and all CFS patients. *p<0.05 for group 1patients with CFS as compared to control (Mann-Whitney U Test).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides methods for diagnosing CFS, together withcell culture procedures for identifying the infectious agent associatedwith CFS. The method for diagnosing CFS involves use of an immunologicprofile at least substantially specific for CFS as compared to theimmunologic profile observed with other conditions such as depression,autoimmunity, and fatigue. To obtain the immunologic profile, peripheralblood mononuclear cell (PMC) subset populations and markers areevaluated for changes relative to those subset populations and markersfound on PMC obtained from a host having symptoms of chronic fatiguesyndrome. The changes associated with a diagnosis of CFS include areduction in CD11b+ cells; an increase in CD11b- cells; and an increasein the percentage of CD8+ cells giving CD38+ and HLA-DR+ markers. All ofthese individual markers are significant (p<0.01) indicators of CFScompared to control populations. Patients with at least 2 of the 3abnormal markers have >90% chance of having CFS.

The following symptoms are associated with chronic fatigue syndrome.Previously, in order to receive a preliminary diagnosis of CSF, apatient needed to fulfill major criteria 1 and 2 and the following minorcriteria: (1) 6 or more of the 11 symptom criteria and 2 or more of the3 physical criteria or (2) 8 or more of the 11 symptom criteria.

Major Criteria

1. New onset of persistent or relapsing, debilitating fatigue in aperson with no previous history of similar symptoms: fatigue that doesnot resolve with bed rest and is severe enough to produce or impairaverage daily activity for a period of at least 6 months.

2. Other clinical conditions that may produce similar symptoms must beexcluded by thorough evaluation, based on history, physical examination,and laboratory findings.

Minor Criteria

Symptom criteria--began at or after onset of fatigue; must havepersisted or recurred over a period of at least 6 months.

1. Mild fever--oral temperature between 37.5° and 38.6° C., if measuredby the patient--or chills.

2. Sore throat.

3. Painful lymph nodes in the anterior or posterior cervical or axillarydistribution.

4. Unexplained generalized muscle weakness.

5. Muscle discomfort or myalgia.

6. Prolonged (24 hours or greater) generalized fatigue after levels ofexercise that would have been easily tolerated in the patient'spremorbid state.

7. Generalized headaches different from ones the patient may have had inthe premorbid state.

8. Migratory arthralgia without joint swelling or redness.

9. Neuropsychologic complaints (e.g. photophobia, transient visualscotomata, forgetfulness, excessive irritability, confusion, difficultythinking, inability to concentrate, depression).

10. Sleep disturbance (hypersomnia or insomnia).

11. Description of the main symptom complex as initially developing overa few hours to a few days.

Physical Criteria

1. Low-grade fever.

2. Nonexudative pharyngitis.

3. Palpable or tender anterior or posterior cervical or axillary lymphnodes.

The present invention greatly simplifies and makes more definite adiagnosis of CFS. The method of the invention for diagnosing CFSinvolves developing a peripheral white blood cell profile of the host(patient) suspected of having CFS. The profile includes identificationof the numbers and types of cells present in a PMC sample obtained fromthe host as well as an evaluation of the percentage of cells expressingactivation antigens. The profile is then analyzed to determine if anyabnormal level (which can be either an increase or a decrease, dependingon the specific marker) of a particular PMC cell subset or marker can befound in comparison to PMC from a healthy control. The profile obtainedwith PMC from a host having symptoms of CFS as compared to PMC from ahost not showing symptoms of CFS (control) is normalized by sample sizeor similar criterion which will allow meaningful comparison of theprofiles obtained for different samples. Conveniently this may beaccomplished by presenting the cell population of a given type in unitsof cells per sample volume, and the relative proportion of such cells asa percentage.

As is typical of diagnostic techniques, a range of differences from themean value of any specific component of the profile is to be expected.Patents with mild symptoms are expected to exhibit only slightdifferences for any of the individual parameters while patents withsevere symptoms are expected to exhibit greater differences. However, aswith any diagnostic assay, different results can occur in patients whodo not respond in the normal fashion of the general population, so thatslight differences can occur in patients with severe symptoms and viceversa. Any of the normal statistical evaluation methods used todetermine the significance of a single value from a mean can be used toevaluate the significance of a given difference. For example, valuesthat fall outside one standard deviation from the mean have about a 90%chance of being statistically different from the mean. Since a range ofmean±one standard deviation would still classify as abnormal relativelylarge portion of the general population, values more than two,preferably at least three, more preferably at least four, standarddeviations from the mean are generally used to evaluate whether a singlevalue (such as percentage of CD8+ cells) is outside the normal range andthus indicative of abnormality (i.e., CFS). However, it should berecognized that the present invention involves testing for the indicatedresults and any statistically significant difference and not necessarilyfinding such differences, since it is probable that a large number ofindividuals test for CFS will be found not to have CFS.

The subset cell profile can be obtained by any of a variety of methodsincluding flow cytometry (FACS); see, for example, Levy et al., Clin.Immunol. Immunopathol. (1985) 35:328. In the FACS method of analysis,monoclonal antibodies to a variety of subset cells bind to and identifyphenotypic antigens present on immune system cells. Commerciallyavailable antibodies exist that can detect the presence of thesemarkers, so that preparation of the antibodies is not required. Onesupplier of these (and other) antibodies is Becton DickinsonImmunocytometry Systems of Mountain View, Calif. Other antibodies whichidentify the same or a closely linked antigenic marker would be expectedto give similar diagnostic results. Thus, where a marker antigen isdesignated in the specification or claims by reference to a particularmonoclonal antibody with which it binds (e.g., CD11b, CD38), such adesignation will be understood to encompass that marker even ifdifferent monoclonal antibodies are used in the identification.Phenotypic markers of interest include general markers for varioussubset cell types including CD3 for total T cells, CD4 for Thelper/inducer cells, CD8 for T suppressor/cytotoxic cells, and CD16/56for NK cells; CD8-expressing subset markers such as CD11b for Tsuppressor cells, CD38 for activated T suppressor/cytotoxic cells,HLA-DR for activated T suppressor/cytotoxic cells, and CD57; and CD4expressing markers such as CD25 and HLA-DR for activated Thelper/inducer cells.

Of particular interest in diagnosing CFS is the evaluation of CD8+cells, particularly for a percentage increase in the subsets of cellsexhibiting activation antigens CD38 and HLA-DR as well as for a decreasein the percentage of CD11b+ cells (or a corresponding increase in CD11b-cells). While it is anticipated that further studies using additionalmonoclonal antibodies against other lymphocyte antigens may revealadditional markers, such additional markers (if found) will not detractfrom the present assay. Thus a diagnosis of CFS can be made byevaluating PMC cells for the markers discussed in this paragraph anddetermining whether one or more statistically different values exist ina given patient (compared to the corresponding values for the generalpopulation). A definitive diagnosis is made by determining a statisticaldifference in at least two of the indicated three values.

The invention also contemplates a kit for diagnosis of chronic fatiguesyndrome comprising a plurality of component monoclonal antibodies orother specific binding molecules, where each of the component antibodiesis specific for one of the cell markers CD11b, CD38, or HLA-DR. Theantibodies are packaged together in a single container or in a pluralityof individual containers for ease of use in the diagnostic assaydescribed above. The antibodies can be bound to a solid support or beprovided in a form suitable for preparation of antibody-containingsolutions. The kit will normally also contain an antibody specific forthe CD8 antigen. Preparation of diagnostic kits for the determination ofspecific antibody binding is well known in the art and need not bedescribed here in detail. The novelty of this aspect of the inventionlies not in preparation per se of the kit but in the selection of thespecific antibodies. Prior to the present invention there has been nopurpose for packaging together the antibodies or other binding moleculesof the specificities described here.

Normal detectable labels can be used for the individual antibodycomponents, such as fluorochromes. When a kit of the invention isdesigned for concurrent use of the antibodies, as occurs in fluorescentcell sorting, each of the component antibodies is labelled so as to beseparately identifiable (e.g., with a fluorochrome that fluoresces at adifferent wavelength).

Methods for identifying and subsequently propagating relatively largeamounts of the causative agent include in vitro tissue culture assaysusing cells susceptible to infection by the agent are also provided bythe invention. Cultures comprising the infectious agent may be obtainedby co-cultivating a human or animal cell culture or cell line with PMCof a CFS patient. Generally the source of infectious agent is a fluidwhich has been in contact with a cell from a host having beenpreliminarily diagnosed as having CFS, which fluid is capable ofinducing CFS-associated changes such as in markers or subset populationsin cultured, normal PMC cells. The cultured cells find use as a sourceof infectious agent (isolate) which may be used directly as a source ofgenomic material for preparing probes for diagnosis, antigens andvaccines, and for post-therapeutic and diagnostic purposes.Agent-specific cells which can be used to culture the CSF agent includePMC cells and selected subsets, particularly NK cells, macrophages, Bcells and CD8+ cells. Various cell lines can also be used, includinghuman cell lines such as a T cell line (for example SUP-T, MT-4),monocyte cell lines (for example U937), B cell lines (for example RAMOS,Raji), fibroblastoid cells (HOS for example), human skin fibroblasts,and neuroblastoma cells (for example SK-N-Mc). Animal cell lines whichfind use include 3T3 (mouse), DHK (hamster), chicken embyro, mink lung,bat lung, NRK (rat kidney), and VERO (monkey).

The general procedures for infecting and culturing the cells with theinfectious agent are as follows. A cultured cell or cell line is placedin contact with a source of agent which is capable of inducing CSFassociated changes in cultured PMC cells, particularly increasing thepercentage of CD8+ cells expressing CD38+ or HLA-DR+. The source can bea fluid obtained directly from a host having symptoms of CFS, such as abodily fluid, particularly whole blood, serum, saliva, and cerebrospinalfluid. Bodily fluid from an animal inoculated with any of the foregoingmaterials, as well as culture fluids (conditioned medium) from forexample, cultures of PMC cells obtained from a CSF host may also beused.

Viral infection of the cells is followed by monitoring for avirus-related cell change over time. Virus infection generally ischaracterized by the appearance of virus-specific antigens, so the viralinfection is properly followed by immunological methods known to thoseskilled in the art (for example, IFA) for detecting antigens. Inaddition, detection may be accomplished by microscopic observation ofcytopathological changes in the inoculated cells, such as the cellballooning and degeneration observed in RAMOS B cells after culture withPMC supernatant from CFS patients.

After viral infection and propagation, the virus can be isolated, ifdesired, by conventional means for releasing and purifying virusparticles from cells. For example, virus particles may be isolated bylysing the cells and subjecting the lysate to standard techniques forfractionating samples containing viruses. Where possible, techniquesthat might disrupt virus particles should be avoided. The isolatedparticles will reproduce the virus-related cell change when cells areexposed to virus particles.

It may be desirable for a variety of reasons to further purify theparticles present in a sample containing particles of the invention. Forexample, if a virus particle is to be treated and employed as a vaccineor in an immunoassay, there ordinarily should be as little in the way ofextraneous protein contamination as possible. Thus, the particle shouldbe substantially free of host proteins.

CFS viral antigens may be obtained from a variety of sources. Theantigen may be present on an intact virus particle, a partially degradedvirus particle, a protein- or carbohydrate-containing molecule insolution, or any other physical form, including an antigen that has beencombined either chemically or physically with a particle or solidsurface, such as by attaching antigens to the surface of a test tube orto suspended particles, such as red blood cells or latex particles. Anantigen of the invention is defined as a substance containing at leastone epitopic site of a virus particle.

To obtain CFS viral antigens, the antigens, whether soluble or in someother form, are typically first separated from cellular contaminants,such as animal cells, cell debris, and cellular microorganisms such asbacteria. This gross separation is generally accomplished bycentrifugation or by filtration using standard techniques. Ordinaryfilters having an average pore diameter of 0.45μ are useful in retaininggross contamination and passing through the antigens.

Additionally, antigens of the invention may be separated from undesiredwater-soluble materials after gross contamination is removed. Where itis desired to recover either intact virus particles or theirwater-insoluble fragments, it is convenient to simply remove all watersoluble constituents from the sample. Suitable techniques includeultrafiltration through a membrane, use of selective flocculating orprotein-precipitating agents (such as polyethylene glycol and ammonitesulfate), and chromatography. Chromatography is the most versatilemethod since it can be readily scaled up for commercial manufacture ofantigen. Gel chromatography systems using cross-linked dextran beads aretypical of the materials used. A column of a suitable gel can beselected which will permit diffusion of proteins and low molecularweight substances into the void volume of the gel beads, therebyretarding the progress of these contaminants through the column, whileallowing whole virus particles to pass through virtually unimpeded. Whena particular antigen is desired, other gel sizes can be selected toprovide for isolation of an antigen of any particular size. The gelwhich is selected will thus be a matter of routine experimentation.

Any of the techniques described herein can be combined as desired. Forexample, isolation of particles on a cesium chloride or sucrose densitygradient can be followed by disruption of particles using any of avariety of techniques and isolation of a viral antigen on gelelectrophoresis, selecting for proteins binding to antibodies, e.g.,antisera, specific for CFS antigens.

One technique that is particularly suitable for isolating solubleprotein antigens or particle fragments is affinity chromatography.Antibodies capable of binding antigens of the invention are covalentlylinked or adsorbed to an insoluble support using conventionalprocedures. The insolubilized antibody is laced in a column. A samplecontaining antigen is passed through the column, where it binds to theinsolubilized antibody. The immunologically-bound antigen is washed withbuffer and can then be released by, for example, changing the ionicstrength or pH of the wash buffer. Generally, an acidic pH is effectivefor releasing the bound antigen. The technique is highly effective inseparating closely related proteins from the antigens of the invention.

Antigens of the invention can be used as a vaccine. A preferred startingmaterial for preparation of a vaccine is the particle antigens producedby tissue culture of the infectious virus. The antigens are preferablyinitially recovered as intact particles as described above. However, itis also possible to prepare a suitable vaccine from particles isolatedfrom other sources or non-particle recombinant antigens. Whennon-particle antigens are used (typically soluble antigens), proteinsnative to the viral envelope are preferred for use in preparingvaccines. These proteins can be purified by affinity chromatography,also described above.

If the purified protein is not immunogenic per se, it can be bound to acarrier to make the protein immunogenic. Carriers include bovine serumalbumin, keyhole limpet cyanin and the like. It is desirable, but notnecessary to purify antigens to be substantially free of human protein.However, it is more important that the antigens be free of proteins,viruses, and other substances not of human origin that may have beenintroduced by way of, or contamination of, the nutrient medium, celllines, tissues, or pathological fluids from which the virus is culturedor obtained.

Vaccination can be conducted in conventional fashion. For example, theantigen, whether a viral particle or a protein, can be used in asuitable diluent such as water, saline, buffered salines, complete orincomplete adjuvants, and the like. The immunogen is administered usingstandard techniques for antibody induction, such as by subcutaneousadministration of physiologically compatible sterile solutionscontaining inactivated or attenuated virus particles or antigens. Animmune response producing amount of virus particles is typicallyadministered per vaccinizing injection, usually in a volume of onemilliliter or less.

In addition to being used in vaccines, the compositions can be used toprepare antibodies to CFS virus particles. The antibodies can beutilized directly as antiviral agents. To prepare antibodies, a hostanimal is immunized using the virus particles or, as appropriate,non-particle antigens native to the virus particle are bound to acarrier as described above for vaccines. The host serum or plasma iscollected following an appropriate time interval to provide acomposition comprising antibodies reactive with the virus particle. Thegamma globulin fraction or the IgG antibodies can be obtained, forexample, by use of saturated ammonium sulfate or DEAE Sephadex, or othertechniques known to those skilled in the art. The antibodies aresubstantially free of many of the adverse side effects which may beassociated with other anti-viral agents such as drugs.

The antibody compositions can be made even more compatible with the hostsystem by minimizing potential adverse immune system responses. This isaccomplished by removing all or a portion of the Fc portion of a foreignspecies antibody or using an antibody of the same species as the hostanimal, for example, the use of antibodies from human/human hybridomas(see below).

The antibodies can also be used as a means of enhancing the immuneresponse since antibody-virus complexes are recognized by macrophages.The antibodies can be administered in amounts similar to those used forother therapeutic administrations of antibody. For example, pooled gammaglobulin is administered at 0.02-0.1 ml/lb body weight during the earlyincubation of other viral diseases such as rabies, measles and hepatitisB to interfere with viral entry into cells. Thus, antibodies reactivewith the CFS virus particle can be passively administered alone or inconjunction with another anti-viral agent to a host infected with a CFSvirus to enhance the immune response and/or the effectiveness of anantiviral drug.

Alternatively, anti-CFS-virus antibodies can be induced by administeringanti-idiotype antibodies as immunogen. Conveniently, a purifiedanti-CFS-virus antibody preparation prepared as described above is usedto induce anti-idiotype antibody in a host animal. The composition isadministered to the host animal in a suitable diluent. Followingadministration, usually repeated administration, the host producesantiidiotype antibody. To eliminate an immunogenic response to the Fcregion, antibodies produced by the same species as the host animal canbe used or the Fc region of the administered antibodies can be removed.Following induction of anti-idiotype antibody in the host animal, serumor plasma is removed to provide an antibody composition. The compositioncan be purified as described above for anti-CFS-virus antibodies, or byaffinity chromatography using anti-CFS-virus antibodies bound to theaffinity matrix. The antiidiotype antibodies produced are specific forCFS-virus particles.

When used as a means of inducing anti-CFS-virus antibodies in a patient,the manner of injecting the antibody is the same as for vaccinationpurposes, namely intramuscularly, intraperitoneally, subcutaneously orthe like in an effective concentration in a physiologically suitablediluent with or without adjuvant. One or more booster injections may bedesirable. The induction of anti-CFS-virus antibodies can alleviateproblems which may be caused by passive administration of anti-CFS-virusantibodies, such as an adverse immune response, and those associatedwith administration of blood products, such as infection.

In addition to therapeutic uses, the particles and antigens of theinvention, as well as the genetic material, can be used in diagnosticassays. Methods for detecting the presence of CFS comprise analyzing abiological sample such as a blood or CFS sample for the presence of ananalyte associated with CFS virus. The analyte can be a nucleotidesequence which hybridizes with a probe comprising a sequence of at leastabout 16 consecutive nucleotides, usually 30 to 200 nucleotides, up tosubstantially the full sequence of a cDNA sequence. The analyte can beRNA or cDNA.

The analyte can be a virus particle having at least one of the followingcharacteristics: obtainable from cells susceptible to infection withCFS, including cells from CFS patients; capable of inducing expressionof virus-specific surface antigen in a cell susceptible to infection bythe particle, the surface antigen being recognized by serum from a hostinfected with CFS and not by serum from a non-infected host; and capableof inducing cytopathological changes in exposed immune cells, such asballooning and degeneration of RAMOS cells. The analyte can comprise anantibody which recognizes an antigen, such as a cell surface viralantigen or a CFS virus particle. The analyte can also be a CFS viralantigen.

In order to detect an analyte, where the analyte hybridizes to a probethe probe may contain a detectable label. Likewise, where the analyte isan antibody or an antigen, either a labelled antigen or antibody,respectively, can be used to bind to the analyte to form animmunological complex, which can then be detected by means of the label.

Typically, methods for detecting analytes such as surface antigensand/or whole particles are based on immunoassays. Immunoassays can beconducted either to determine the presence of antibodies in the hostthat have arisen from infection by CFS virus or by assays that directlydetermine the presence of virus particles or antigens. Such techniquesare well known and need not be described here in detail. Examplesinclude both heterogeneous and homogeneous immunoassay techniques andare based on the formation of an immunological complex between the virusparticle or its antigen and a corresponding specific antibody.Heterogeneous assays for viral antigens typically use a specificmonoclonal or polyclonal antibody bound to a solid surface. Sandwichassays are becoming increasingly popular. Homogeneous assays, which arecarried out in solution without the presence of a solid phase, can alsobe used, for example by determining the difference in enzyme activitybrought on by binding of free antibody to an enzyme-antigen conjugate.

When assaying for the presence of antibodies induced by CFS viruses, theviruses and antigens of the invention can be used as specific bindingagents to detect either IgG or IgM antibodies. Since IgM antibodies aretypically the first antibodies that appear during the course of aninfection, when Ig synthesis may not yet have been initiated,specifically distinguishing between IgM and IgG antibodies present inthe blood stream of a host will enable a physician or other investigatorto determine whether the infection is recent or chronic. Thisinformation can then be used in conjunction with the immunologicalprofile, particularly where the immunological profile is abnormal butnot significantly different from normal. For example, if only IgMantibodies are present, it may be useful to reevaluate the host at3-month intervals.

The genetic material of the invention can itself be used in numerousassays as probes for genetic material present in naturally occurringinfections. It may be amplified as necessary. One method foramplification of target nucleic acids, for later analysis byhybridization assays, is known as the polymerase chain reaction or PCRtechnique. The PCR technique can be applied to detecting virus particlesof the invention in suspected pathological samples using oligonucleotideprimers. The PCR method is described in a number of publications,including Saiki et al., Science (1985) 230:1350-1354; Saiki et al.,Nature (1986) 324:163-166; and Scharf et al., Science (1986)233:1076-1078. Also see U.S. Pat. Nos. 4,683,194; 4,683,195; and4,683,202.

For both in vivo use of antibodies to CFS-virus particles and proteinsand anti-idiotype antibodies and diagnostic use, it may be preferable touse monoclonal antibodies. Monoclonal anti-virus particles antibodies oranti-idiotype antibodies can be produced as follows. The spleen orlymphocytes from an immunized animal are removed and immortalized orused to prepare hybridomas by methods known to those skilled in the art.To produce a human-human hybridoma, a human lymphocyte donor isselected. A donor known to be infected with a CFS virus (where infectionhas been shown for example by the presence of anti-virus antibodies inthe blood or by virus culture) may serve as a suitable lymphocyte donor.Lymphocytes can be isolated from a peripheral blood sample or spleencells may be used if the donor is subject to splenectomy. Epstein-Barrvirus (EBV) can be used to immortalize human lymphocytes or a humanfusion partner can be used to produce human-human hybridomas. Primary invitro immunization with peptides can also be used in the generation ofhuman monoclonal antibodies.

Antibodies secreted by the immortalized cells are screened to determinethe clones that secrete antibodies of the desired specificity. Formonoclonal anti-virus particle antibodies, the antibodies must bind toCFS virus particles. For monoclonal antiidiotype antibodies, theantibodies must bind to anti-virus particle antibodies. Cells producingantibodies of the desired specificity are selected.

The following examples are offered by way of illustration and not bylimitation.

EXAMPLE 1 Immunologic Profile of Patients With CFS Subjects

A total of 147 consecutive patients (30 males and 117 females, age range15-80, median age 38) who were presented for evaluation as having CFSwere studied from September 1989 to November 1990. As determined eitherat the time of evaluation or by past medical history, their illness hadbeen present for 1-3 years. All patients met the criteria for CFS asdefined by the CDC (Kruesi M., J. Clin. Psychiatry (1989), 50(2):53-6)and in Table I below.

                  TABLE I                                                         ______________________________________                                        Symptoms of Patients with CFS                                                              Group 1.sup.b                                                                          Group 2.sup.c                                                                          Total CFS Patients                             Symptoms     (n = 67) (n = 21) (n = 47)                                       ______________________________________                                        Exhaustion/Fatigue                                                                         100.sup.a                                                                              5        70                                             Post exertional weak-                                                                      100      24       92                                             ness                                                                          Muscle Weakness                                                                            96       0        56                                             Severe Cognitive                                                                           92       0        74                                             Dysfunction.sup.d                                                             Abdominal/Gastro-                                                                          90       0        84                                             intestinal Distress                                                           Nausea       89       0        46                                             Neuroirritability                                                                          89       5        69                                             Sleep Disorder.sup.e                                                                       89       0        58                                             Twitching/Myoclonus                                                                        89       10       56                                             Frequent Headache                                                                          81       14       59                                             Balance problems                                                                           75       5        66                                             Depression.sup.f                                                                           73       0        46                                             Chills       55       0        33                                             Sore throat  33       5        20                                             Lymph Node Pain                                                                            30       0        19                                             ______________________________________                                         .sup.a Figures give percent of patients exhibiting symptoms.                  .sup.b Patients in this group have 25% or less normal physical activity       and major CFS symptoms.                                                       .sup.c Patients in this group have 75% or more normal physical activity       and mild CFS symptoms. They had marked improvement for at least 2 months      in their clinical condition at the time of study.                             .sup.d Short term memory loss, encoding, stimulus recognition.                .sup.e Hypersomnia and hyposomnia.                                            .sup.f In the majority of cases, the onset of depression occurred 6 month     after the onset of the illness.                                          

Control subjects consisted of healthy individuals (n=50) living in SanFrancisco and working at the UCSF Medical Center and subjects (n=30)seeking medical attention for clinical conditions other than CFS. Themajority of these other individuals were being seen for routine physicalexaminations. Immunophenotypic data on these two control groups were notsignificantly different, so they were combined for the study as onecontrol group (30 males, 50 females, age range 20-55, median age 32).Additional control populations evaluated and spouses and family membersof CSF patients (n=11); medical personnel in contact with CFS patients(n=11); patients with acute viral-like illness (n=15); patients withsystemic lupus erythematosus (SLE) (n=12) as defined by the AmericanRheumatology Association; patients with documented depression (n=10);and individuals with prolonged fatigue without any other clinicalcriteria for CFS (n=6). All non-CFS subjects were referred by physiciansin the San Francisco and Chicago areas.

Blood Samples

EDTA anticoagulated blood was collected for flow cytometric studies,white blood cell counts, and differential counts. An additional serumsample was obtained for viral serologic studies.

Viral Serology

Quantitative serology was done to measure the levels of antibodies tovarious viruses in randomly selected sera from 40 healthy laboratorypersonnel controls compared to those obtained with 63 CFS patientsrepresenting 23 individuals in group 1 and 27 individuals in group 2 (asdefined in Table I). The viral agents evaluated included those proposedas "candidate" agents for CFS as well as other viruses. Antibody toAdenovirus, Coxsackie B4, human herpes virus 6 (HHV6), human T cellleukemia virus I/II (HTLV-I/II), human immunodeficiency virus (HIV),rubeola, and papovavirus were assayed by indirect immunofluorescence(Lennette, J. Clin Microbiol. (1987) 25:199-200); antibody response tocytomegalovirus was evaluated by immune adherence hemagglutination (CMV)Lennette, J. Clin. Microbiol. (1978) 7:282-85); and Epstein-Barr virus(EBV) early antigen (EA), viral capsid antigen (VCA) and nuclear antigen(EBNA) were measured by both indirect immunofluorescence andanti-complement indirect immunofluorescence (Reedman, Int. J. Cancer(1973) 11:499-520; and Lennette (1987) supra).

Sample Preparation and Flow Cytometric Analysis

Lymphocyte and monocyte populations were analyzed by flow cytometryusing dual color direct immunofluorescence after whole blood lysis (Coonet al., Lab. Invest (1987) 57:453-79). The panels of fluoresceinisothiocyanate (FITC) or phycoerythrin (PE) monoclonal antibodies usedare listed in Table II below. A single laser flow cytometer (FACScan™):Becton Dickinson Immunocytometry Systems, San Jose, Calif.) which hasthe capability of discriminating forward and side scatter as well as twofluorochromes was used with the Consort 30 software.

                  TABLE II                                                        ______________________________________                                        Immunophenotyping Panel Used for Evaluation of CFS Patients                   Cell Subset   Antibody Specificity                                            ______________________________________                                        CD3           Total T                                                         CD4           T helper/inducer (T H/I)                                        CD3CD8        Cd8.sup.+  T suppressor/cytotoxic (T S/C)                       CD8CD11b      Suppressor T cell                                               CD8CD28       Cytotoxic T cell                                                CDUCD57       T S/C Subset                                                    CD8Leu8       T S/C Subset                                                    CD16CD56      Natural killer                                                  CD20          B cell                                                          CD5 CD20      B cell subset                                                   CD14          Monocyte                                                        Activation Markers                                                            CD4CD25       Activated T H/I                                                 CD4HLA-DR     Activated T H/I                                                 CD8CD38       Activated T S/C                                                 CD8HLA-DR     Activated T S/C                                                 CD8CD25       Activated T S/C                                                 CD16CD25      Activated NK                                                    CD16HLA-DR    Activated NK                                                    CD14CD25      Activated NK                                                    Cell Adhesion Markers                                                         CD11a         Adhesion molecule integrin family                                             (LFA1-a)                                                        CD18          Adhesion molecule integrin family                                             9LFA1-b)                                                        CD44          Homing receptor                                                 CD54          Intracellular adhesion molecule                                               (ICAM-1)                                                        ______________________________________                                    

The method used was as follows. A 100 μl sample of whole blood wasaliquoted into tubes, monoclonal antibody was added, and the mixtureincubated for 15 minutes at room temperature. Red cells were lysed witha commercial lysing reagent (FACsLyse™) for 10 minutes, and the lysatespun down and washed with phosphate buffered saline (PBS) containing 3%fetal calf serum and 0.1% sodium azide. The white cells were resuspendedin the wash buffer and fixed with a final concentration of 1%paraformaldehyde. Lymphocyte gates were confirmed by the HLE FITC(CD45), LeuM3 PE (CD14) antibody combination (Leukegate). Evaluation ofthe cell adhesion antigens on lymphocyte, monocyte, and neutrophilpopulations was performed using indirect immunofluorescence staining.

Data Analysis and Statistics

The Mann-Whitney U and Kruskall-Wallis tests were used to evaluatedifference in immunologic markers between the groups of patients, sincethese markers are known to have non-Gaussian distribution (Siegel,(1956) McGraw Hill Book Co., New York). Student's T-test for independentsamples or one-way analysis of variance (SPSS-PC⁺ Release 4.0, SPSS,Inc., Chicago) was used to evaluate differences between the groups forother continuous variables. When more than 2 groups were evaluated, theScheffe' test was employed to identify the groups for which thedifferences were significant.

Clinical Evaluation of CFS Patients

The individuals referred with the suggested diagnosis of CFS wereevaluated for a large number of symptoms (Table I), past medical historyand physical findings. A full report on the clinical aspects of theirillness will be the subject of another publication. All the patients hada past history of fatigue for more than six months, post exertionalweakness substantially worse than previously observed in the past,muscle weakness, myalgias and frequent headaches. Many hadgastrointestinal symptoms with nausea, and neurologic findings thatinclude sleep disorders, severe cognitive dysfunction,neuroirritability, myoclonus and balance problems (Table I). About 50%gave a medical history of flu-like illness at the onset of this clinicalcondition. Only 8 patients had depression prior to the onset of theillness, but many (80%) developed depression after 2 years of illness.Based on their symptomatology, the CFS patients could be placed intothree groups: Group 1 consisted of 67 patients whose illness was sosevere that they had less than 25% of their normal daily activity andmultiple symptoms, Group 2 consisted of 21 patients who initially hadsevere symptoms and incapacitating illness like Group 1; however, at thetime of evaluation they had made substantial improvement for at least 2months and regained 75% or ore of their normal physical activity withonly mild symptoms; 59 individuals with reduced physical activitycontinued to have moderate symptoms. Healthy controls had none of thesymptoms listed in Table I on a persistent basis.

Viral Serology

The prevalence and titers of antibodies to CMV, EBV-VCA, EBNA, rubeola,adenovirus type 2, and the papovavirus (BK virus) did not differ betweenhealthy controls and CFS patients (Table III).

                  TABLE III                                                       ______________________________________                                        Comparative Serosurvey of Antiviral Antibodies                                in CFS Patients and Control Subjects                                                     Healthy Controls                                                                          CFS Patients                                                      N = 40      N = 63                                                 Virus        GMT.sup.a                                                                             (%+)      GMT   (%+)                                     ______________________________________                                        Cytomegalovirus                                                                             42     (40)       67   (46)                                     Epstein-Barr                                                                  VCA          314     (95)      393   (95)                                     EA            25     (15)       40   .sup. (51).sup.b                         EBNA         206     (95)       96   (95)                                     Rubeola      473     (98)      577   (89)                                     HHV-6        104     (98)      201   (100)                                    Adenovirus   285     ((%)      155   (81)                                     Coxsackie B4 113     (65)      134   .sup. (90).sup.b                         Papovavirus BK                                                                              83     (55)       61   (44)                                     HTLV-II               (0)             (0)                                     HIV-1/HIV-2           (0)             (0)                                     ______________________________________                                         .sup.a GMT = geometric mean titer.                                            .sup.b The seroprevalence is statistically significant (p<0.001) by Chi       square analysis.                                                         

Evaluation of CFS patients based on clinical status as defined in TableI also showed no differences in these viral antibody titers as comparedto controls. No antibodies to HTLV-I/II and HIV were found in anysubjects. Antibody titers to HHV-6 in the CFS patients were twice ashigh as those found in controls, but were not significantly different(p>0.05). The prevalence of antibodies to Coxsackie B4 virus wassignificantly higher in the CFS group compared to controls (90% vs. 65%)(p<0.001), but the geometric mean titers were approximately the same.Moreover the prevalence of antibodies to EBV-EA was significantlydifferent between the CFS patients and controls 51% vs. 15%) (p<0.001).This finding was specific for EA since the VCA and EBNA seropositivityrates for both groups were identical.

Peripheral white Blood Cell Analyses: Lymphocyte nd Monocyte PhenotypicProfile

The total number of white blood cells (6-10×10³ /mm³) was not differentbetween CFS patients and healthy control groups. Evaluation of the totallymphocyte, monocyte and neutrophil populations also showed nodifferences between control and CFS patients whether the latter wereconsidered as total or separate groups (Table I). Cell surfacephenotypic analysis of the major lymphocyte populations including T(CD3), B (CD20), and NK (CD16+/CD56+) cells indicated no significantdifferences between CFS patients and controls (Table IV).

                  TABLE IV                                                        ______________________________________                                        Peripheral Blood Phenotypic Profile in CFS                                    and Healthy Individuals                                                                          CFS       Controls                                         Surface Markers    (n = 147) (n = 80)                                         ______________________________________                                        % CD3.sup.+  (total T cell)                                                                      71 ± 8.sup.a                                                                         74 ± 8                                        % CD4.sup.+  cell  46 ± 9 48 ± 9                                        (T helper/inducer cell)                                                       CD4.sup.+  cell number                                                                           834 ± 315                                                                            889 ± 273                                     % CD8.sup.+  cell  24 ± 8 22 ± 4                                        (T suppressor/cytotoxic)                                                      CD8.sup.+  cell number                                                                           468 ± 227                                                                            488 ± 220                                     % CD20.sup.+  cell (B cells)                                                                     11 ± 16                                                                              10 ± 3                                        % CD16/CD56.sup.+  (NK cell)                                                                     13 ± 4 14 ± 8                                        Helper/Suppressor Ratio                                                                          1.7 ± 0.5                                                                            1.8 ± 0.4                                     % CD4+ Cells Expressing                                                       CD25               33 ± 7 34 ± 6                                        HLA-DR             12 ± 6 10 ± 7                                        % CD16+ Cells Expressing                                                      CD25               4 ± 1  3 ± 2                                         HLA-DR             3 ± 1  3 ± 1                                         % CD14+ Cells Expressing                                                      CD25               >1        >1                                               ______________________________________                                         .sup.a Data represent mean ± standard deviation.                      

Evaluation of the percentage and absolute number of CD4⁺ and CD8⁺ Tcells also demonstrated no significant differences (p>0.05) from thecontrols (Table IV). Analysis of the CD4:CD8 ratios of cells in ourpatient population showed 88% had ratios within our normal range(1-2.5), 5% had decreased CD8⁺ cells and 7 had increased CD8⁺ cells(ratio<1.0). Taken together the average CD4/CD8 ratio for all CFSpatients (1.7+0.5) did not differ significantly from controls (1.8+0.4)(p>0.05).

Analysis of activation antigens (CD25 and HLA-DR) on CD4⁺ T cells, NKcells or monocytes showed no significant differences in CFS patientsfrom healthy controls. Analysis of a B cell subset (CD5/CD20) that hasbeen found to be elevated in autoimmune disease (Casali et al., Science(1987), 236:77-81) also indicated no differences between CFS and healthycontrols. In addition, flow cytometric studies of the cell adhesionantigens (CD11a, CD18, CD44, CD54) showed no differences in percentagepositive or the fluorescence intensity of cells obtained from CFSpatients compared to healthy controls.

CD8 Cell Subset Analysis

Previous cell surface marker studies in documented acute viralinfections such as EBV, CMV, and HIV infection, have shown elevation ofCD8⁺ cells which express activation antigens (CD38, HLA-DR) (Carney etal., J. Immunol., 1981, 126:2114-16; Tomkinson et al., J. Immunol.,1987, 139:3802-07 and Landay et al., Sixth International Conference onAIDS, San Francisco, Calif., 1990, p. 141). In the case of the herpesviruses, these cell numbers return to normal 2 to 4 weeks followinginfection. We evaluated a number of cell surface antigens expressed onCD8⁺ T cells from the 147 CFS patients and compared them to the 80healthy controls (FIG. 1). In addition, the results with the twoseparate clinical subgroups (Table I) were considered.

Three markers gave noteworthy results. With the total CFS patients, thepopulation of CD8⁺ cells expressing CD11b were somewhat decreased whencompared to the normal controls (19±16 vs. 25±10) indicating a drop inthe phenotypic suppressor CD8⁺ T cell compartment. Since the number oftotal CD8⁺ cells is not changed, a concomitant increase in thephenotypic cytotoxic (CD8⁺ CD28⁺ CD11b-) population is seen. This resultwas confirmed in a preliminary study showing a rise in the CD8⁺ CD28⁺population in these patients. Evaluation of activation antigens on theCD8⁺ cells showed an increase in CD38 (47±20 vs 35±12 for Controls) andHLA-DR (22±8 vs 14±6 for controls) expression. An additional activationantigen CD25, as well as the CD57 and Leu8 antigens were expressed onthe CD8⁺ cells at a level comparable to controls.

When these same CD8⁺ cell subsets were considered in Group 1 patients,the differences as compared to healthy controls (FIG. 1) reachedstatistical significance. In contrast, evaluation of the CD8⁺ cellsubsets among the CFS patients who improved in their clinical status(Group 2) showed no significant differences from healthy controls (FIG.1). The 59 patients who had moderate symptoms also had CD8⁺ cellabnormalities, but they were not statistically significant. Furtherevaluation of Group 2 showed only 10% of patients had two or moresignificantly abnormal results among the CD8⁺ CD11b⁻, CD8⁺ CD38⁺, or CD8+HLADR⁺ subsets, whereas among the Group 1 patients 85% had 2 or moreabnormal results. These data indicate a high probability (90%) of havingactive CFS if an individual has two or more of the CD8⁺ cell subsetalterations.

To control for possible alteration in cell surface antigens by the wholeblood lysis procedure, a subset of patients was evaluated followingFicoll-Hypaque isolation of peripheral blood mononuclear cells. Nodifferences were seen between the whole blood lysis procedure andFicoll-Hypaque purified cells.

CD8 Cell Markers in Other Control Populations

To evaluate the specificity of the CD8⁺ cell alteration in CFS patients,several individuals with other clinical conditions were evaluated withthe same panel of monoclonal reagents. Among patients who had an acuteviral-like illness (common cold or flu-like illness), a number ofobservations were made. The initial cellular response demonstrated anincrease in both percentage and absolute numbers of natural killer cells(CD16⁺ /CD56⁺). These natural killer cells were CD8⁺, CD38⁺, CD11b⁺, andHLADR⁻ as determined by multiparameter flow cytometric studies. Inseveral of the individuals studied we noted this initial natural killerresponse was followed by an increase in activated CD8/CD11b T cells(CD38⁺ HLA-DR+). In all these subjects, recovery 1-2 weeks later wasaccompanied by a return to normal of all these immunologic parameters.All other CD8⁺ cell markers were normal in these individuals.

Evaluation of CD8⁺ subsets in patients with a diagnosis of depressionshowed no significant differences compared to healthy controls.Furthermore, family members and contacts of CFS patients demonstratednormal CD8⁺ subsets as well as those individuals presenting with fatigueother than CFS (Table V).

                                      TABLE V                                     __________________________________________________________________________    CD8.sup.+  Cell Surface Markers in Control Populations                                                      Chronic                                                Acute                                                                              Autoimmune                                                                           Family     Fatigue                                                                             Autoimmune                                                                           Healthy                            CD8.sup.+  Cells                                                                     Viral                                                                              Depression                                                                           Members.sup.b                                                                       Contacts.sup.c                                                                     CF Alone                                                                            Disease                                                                              Controls                           Expressing                                                                           (n = 15)                                                                           (n = 10)                                                                             (n = 11)                                                                            (n = 11)                                                                           (n - 6)                                                                             (n = 12)                                                                             (n - 80)                           __________________________________________________________________________    CD11b  42; 12                                                                              28; 10                                                                               27; 12                                                                             27; 8                                                                               30; 12                                                                             28; 6  25; 10                             CD38   .sup. 63; 10.sup.a                                                                 26; 8  28; 7 32; 6                                                                              25; 6  45; 12                                                                              53; 12                             HLA-DR 12; 3                                                                              10; 5  10; 6 12; 6                                                                               9; 3 15; 4  14; 6                              CD16/56                                                                              24; 7                                                                              12; 4   8; 2 11; 4                                                                              10; 5 12; 6  14; 8                              __________________________________________________________________________     .sup.a Statistically significant (Schiffe test) compared to healthy           controls at p<0.05.                                                           .sup.b Spouses or family members of CFS patients.                             .sup.c Contacts are medical personnel coming in contact with CFS patients                                                                              

Laboratory findings among CFS patients have shown low levelautoantibodies which may reflect an underlying autoimmune disease(Buchwald et al., Review Infect. Dis., 1991, 13:512-18). Evaluation ofpatients with SLE showed only increased expression of the CD38 marker onCD8⁺ cells (Table V); other cell surface markers were within the normalrange.

Example 2 Isolation of Infectious Agent for CFS

1. Inoculation of Animals

One method for identifying a new agent involved in human disease is toinfect a susceptible animal with putatively diseased tissue. The animalscan be observed for the development of symptoms, and the agent can bedetected by standard microbiological and serological methods. We usenewborn NSF mice selected because of their low level endogenous virusproduction (Levy Current Topics in Microbiology and Immunology (1978)7:111), and the sensitivity of mice to a variety of human agents(Hsiung, Diagnostic Virology (1982); and Prusiner, Ann. Rev. Micro.(1989) 43:345). Syrian hamsters are chosen for their susceptibility toseveral infectious agents including human parvoviruses and "prions,"(Fraenkel-Conrat, Virology (1988); Prusiner, Ann. Rev. Micro. (1989)43:345; and Berns, Plenum Press (1984)), their relative ease of study,and low cost of purchase and maintenance. After inoculation with patientmaterial, each animal is observed for development of physicalabnormalities or disease. The animals are inoculated intraperitoneally(IP) or subcutaneously (SC) with the following patient specimens:

a. 0.1-0.2 ml whole blood

b. separated PMC (10³⁻¹⁰ ⁶ cells) in 0.2 ml

Control animals receive blood and PMC from normal, healthy donors. Inaddition, some hamsters receive material intracerebrally, since thisapproach has helped detect the presence of human parvoviruses and prions(Fraenkel-Conrat, Virology (1988); Prusiner, Ann. Rev. Micro. (1989)43:345; and Berns, K.I. Plenum Press (1984).

All specimens are inoculated within 24 hrs. after removal from thesubject. The PMC of the CFS patient are separated by Ficoll/Hypaquedensity gradient as is routinely conducted in the laboratory (Castro, J.Clin. Microbiol. (1988) 26:2371). The PMC are suspended in RPMI 1640medium without serum for inoculation into the animals. Similar studieswith small animals were conducted in the initial attempts to find theAIDS virus, (Morrow, J. Gen. Virol. (1987) 68:2253).

For these studies, the specimens from 10 CFS patients are injected into100 newborn animals from each species each year. These animals arefollowed for any signs of disease, including neurologic abnormalities orchanges in their immune responses (see below). Litter mates receiveblood or PMC from 5 normal donors as controls.

2. Studies on Inoculated Animals

a. The animals are followed daily for any signs of disease. A fullphysical examination is conducted weekly. They are evaluated for failureto thrive, ruffled fur, enlargement of the spleen or thymus, pneumoniaand neurologic disorders (unsteady gait or paralysis).

b. For the first three months, control animals and those inoculated withpatient material are bled once every two weeks either intraorbitally orfrom the tail vein. A complete blood count and differential is done. Inparticular, activation markers on the CD8+ cells of mice (for whichmarkers are available) are measured (Eichmann, Immunol Rev. (1989)109:39). In hamsters, the relative presence of lymphocytes, andmacrophages is assessed and compared to controls. Serum is collectedmonthly to test for a potential agent that has hemagglutinating activityagainst human, chick, goose, guinea pig and sheep erythrocytes asmeasured by conventional techniques (White, Medical Virology (1986) andHsiung, Diagnostic Virology (1982)). These approaches were used in theinitial search for the AIDS virus. After three months, these studies arerepeated every 6 weeks or when animals show signs of disease. Allanimals are maintained for one year.

c. Any sick animal or animal showing a change in normal immunologicprofile is sacrificed and evaluated using the following protocol:tissues are examined for gross pathology and fixed for microscopicexamination (see Table A). Any tissues showing abnormalities areexamined by electron microscopy (EM) (see Table B). Extracts of thetissues and the blood are reinoculated into newborn animals of the samestrain in an attempt to transfer the putative etiologic agent. They arealso inoculated onto selected cultured animal and human cells that areexamined for evidence of a pathogen such as CPE, hemadsorption, RTactivity (see Table 6). With some sick animals, attempts are made toestablish the animal cells, particularly PMC, in culture for furtherevaluation.

d. The PMC of the mouse strains separated by Ficoll/Hypaque proceduresare grown in the presence of mouse T cell growth factor (Smith, Immunol.Rev. (1980) 51:337), which is available commercially. Human IL-2 alsocan be used for these procedures as well as those using separated PMCfrom hamsters. Supernatants from inoculated cells and cell lines arechecked for reverse transcriptase activity (Mn++ and Mg++ dependent) andhemagglutinating activity (White, Medical Virology (1986) and Hsiung,Diagnostic Virology (1982). If positive, they are inoculated back intoanimals to assess the presence of a pathologic agent.

e. Sera from the animals, after absorption with normal humanlymphocytes, is tested by immunofluorescence assays (IFA) for reactivitywith PMC cultures of individuals with CFS and with cells inoculated withspecimens from these animals. Any positive result may reflect growth ofthe agent in the animal host.

3. Tissue Culture Studies

a. Whole blood, PMC, sera, saliva and cerebrospinal fluid specimens frompatients with CFS are evaluated for the presence of an infectious agentby inoculation onto normal PMC cultures and a variety of cell lines (seeTable VI), following pretreatment with polycations to increase theirsensitivity to virus infection (Castro, J. Clin. Microbiol. (1988)26:2371).

                  TABLE VI                                                        ______________________________________                                        Cell Lines Used for Detection of the CFS Agent*                               Animal Cell Lines                                                                              Human Cells                                                  ______________________________________                                        mouse 3T3        Sup-T - T cell line                                          BHK hamster      MT-4 - T cell line                                           chicken embryo   U937 - monocyte cell line                                    mink lung        RAMOS - B cell line                                          bat lung         Raji - B cell line                                           NRK - (rat) kidney                                                                             HOS - human fibroblastoid                                    Vero (monkey)    cells                                                                         human skin fibroblast                                                         SK-N-Mc - neuroblastoma                                                       cells                                                        ______________________________________                                         *Cell lines are routinely checked for mycoplasma contamination. These         lines are selected because of their cell type and use in the laboratory       for growth of known viruses.                                             

These cells are subsequently examined for an infectious agent by theprocedures listed in Table VII, including hemadsorption procedures usingguinea pig and chick erythrocytes (White, Medical Virology (1986) andHsiung, Diagnostic Virology (1982)). This latter approach wasinstrumental in initially finding the simian type D retrovirus (Marx,Science (1984) 223:1083).

                  TABLE VII                                                       ______________________________________                                        Procedures to be Used Routinely for Detection of an                           Infectious Agent in Cultures of CFS Tissues                                   ______________________________________                                        1.   Cytopathic effects (balloon degeneration, enlarged cells,                     syncytia)                                                                2.   Hemadsorption (cells)                                                    3.   Hemagglutination (fluid)                                                 4.   Reverse transcriptase activity (Mg.sup.++, Mn.sup.++) (fluid)            5.   Induction of antigens in the cells (detected by IFA)                     ______________________________________                                    

Supernatant from these cultures is passed twice to fresh cells of thesame type to enhance the sensitivity for detection of the agent.Endogenous retroviruses can be expressed in some of the cell lines(e.g., mouse, rat, mink, MT-4) (Levy, Cancer Res. (1986) 46:5457 andLevy, Current Topics in Microbiol. and Immunol. (1978) 7:111) and any RTactivity is assessed for its association with an endogenous virus.Specific antisera against these agents are available.

4. Isolation of an infectious agent

Our laboratory, over the past four years, has evaluated peripheral bloodmononuclear cells (PMC), cerebrospinal fluid (CSF), saliva, and cervicalswabs obtained from 96 patients with CFS for the presence of aninfectious agent (see Table VIII).

                  TABLE VIII                                                      ______________________________________                                        Tissue Culture Studies to Detect Infectious Agents in                         Chronic Fatigue Syndrome                                                      Specimen       No. Tested                                                                              No. Positive#                                        ______________________________________                                        PMC            96        8                                                    Plasma         24        1                                                    CSF             2        0                                                    Saliva         15        1                                                    Cervical swab   1        0                                                    ______________________________________                                         #See Table 4 for details on positive specimens.                          

These studies involved the culturing of the PMC with and withoutmitogen-stimulated normal PMC similar to approaches used to isolate theAIDS virus (Castro, et al., J. Clin. Microbiol. (1988) 26:2371). Inaddition, in some studies, the isolation of a potential agent wasattempted on a variety of different animal and human cell lines (seeTable VII). Thus far, evidence of a potential cytopathic agent has beennoted in eight of the individuals studied (Table VIII). Cytopathologywas observed in the PMC cultures of three, and in three others whenfluid from the PMC culture was passed to the RAMOS B cell line (seeTable IX).

                  TABLE IX                                                        ______________________________________                                        Detection of Infectious Agents Associated with Chronic                        Fatigue Syndrome                                                              Specimens    Cells Detected                                                                            How Detected                                         ______________________________________                                        PMC          PMC         CPE                                                  PMC          PMC         CPE                                                  PMC          PMC         CPE                                                  PMC          PMC         RT                                                   PMC          PMC         RT                                                   PMC          PMC         RT                                                   PMC          Ramos       CPE                                                  PMC          Ramos       CPE                                                  PMC          Ramos       CPE                                                  Saliva*      PMC         CPE                                                  Plasma*      PMC         CPE                                                  ______________________________________                                         RT  Mn++-dependent reverse transcriptase activity in the culture fluid.       CPE  cytopathic effects as demonstrated by giant cells and balloon            degeneration in the cells listed (FIG. 1).                                    PMC  peripheral blood mononuclear cells.                                      *PMC of patient also gave evidence of an infectious agent.               

In 1 out of 15 patients, saliva induced cytopathic changes in the PMCculture. Neither of 2 cerebrospinal fluids nor a single cervicalspecimen showed any CPE-inducing agent, but one out of fifteen plasmasamples gave CPE in RAMOS cells. The observations with saliva were mostlikely due to HHV-6 as this virus has recently been found in the salivaof most individuals (Levy, et al., The Lancet (1990) 335:1047). None ofthe animal or other human cells showed evidence of any agent bymorphologic or serologic criteria (e.g., IFA).

As noted above, in six PMC cultures an agent was found that led tocytopathic changes in the RAMOS B cell line. Subsequent studies usingimmunofluorescent techniques (conducted by E. Lennette) indicated thatthis CPE was associated with staining of 0.1% of the cells by CFS sera;the results did not reflect infection by EBV, HHV-6 or any other humanvirus considered (see Table X for list).

                  TABLE X                                                         ______________________________________                                        Antibodies to Human Viruses in CFS Patients and Controls                                CFS         Control                                                 Viral Agent      Positive          Positive                                   ______________________________________                                        Adenovirus type 2                                                                         N = 64   19 (30%) N = 10 5 (50%)                                  Coxsackie B4                                                                              N = 64   35 (55%) N = 10 6 (60%)                                  N = 64                                                                        CMV         N = 92   45 (49%) N = 10 5 (50%)                                  Measles     N = 28   26 (89%) N = 28 26 (92%)                                 EBV         N = 64   63 (89%) N = 10 9 (90%)                                  HHV-6       N = 52   51 (98%) N = 59 58 (99%)                                 HIV         N = 96   0 (0%)   N = 59 0 (0%)                                   HTLV-I/II   N = 64   0 (0%)   N = 10 0 (0%)                                   ______________________________________                                    

Subsequent passage of supernatant from the cultures to fresh RAMOS cellsdid not lead to the isolation of an infectious agent. The induction ofCPE and antigen was achieved for 2-3 passages and then these cellculture observations were no longer observed. In the final cultures inwhich the virus was not noted, attempts to activate the agent withhalogenated pyrimidines (e.g., IUDR) also did not recover a CPE-inducingagent.

The fluid from one PMC culture induced cytopathic changes leading tocell death in macrophage cultures from normal controls (Table VIII).Passage of this filterable CPE-producing agent in the supernatant wasachieved for 2 passages and then lost as was our experience with theRAMOS cell agents described above.

Finally, in 31 PMC cultures evaluated for reverse transcriptase (RT)activity (Hoffman, et al., Virology (1985) 147:326), 3 cultures gavelevels >10,000 cpm/ml of culture supernatant using Mn⁺⁺ as the cation.No RT activity with Mg⁺⁺ was noted. For these studies, we routinely usedfrozen and thawed cell pellets to detect any cell-associated viruses asis the case with HTLV-1 (Fraenkel-Conrat, et al., Virology 2nd Ed.(1988); and Levy, Cancer Res. (1986) 46:5457). However passage of the RTactivity to fresh cultures did not lead to induction of RT activity norisolation of a retrovirus. We have presumed the initial RT activityreflected cellular polymerases.

b. PMC from CFS patients is cultured in RPMI 1640 medium with andwithout IL-2, with and without PHA, and cocultivated with PMC fromnormal donors (obtained from Irwin Memorial Blood Bank, San Francisco)or the Ramos cell line. (Castro, J. Clin. Microbiol. (1988) 26:2371).These approaches permit relative preferential growth of white cellsubsets that may or may not yield the CFS agent. (Castro, J. Clin.Microbiol. (1988) 26:2371). The Ramos line is used because of itsprevious signs of CPE after inoculation with PMC cell fluid from a CFSpatient (see FIG. 1). These cultures are examined for CPE and RTactivity, and other signs of an infectious agent (see Table 6) includinginduction of antigens detected by IFA using the patient's serum. Fluidis also inoculated onto selected animal and human cell lines (see TableVI) for further evaluation of a replicating agent (see FIG. 2 forsummary of this approach).

c. Sera from individual patients is used to examine the cells by IFA fora potential infectious agent.

d. Sera from individual patients is also examined for the presence of aninfectious agent by measuring hemagglutinating activity usingconventional assays with sheep, chick, guinea pig, and humanerythrocytes (White, Medical Virology (1986)).

e. Culture of Purified subsets of peripheral white cells obtained fromCFS patients

1) Attempts will be made to identify the CFS infectious agent in cultureby techniques developed for efficient isolation of the AIDS virus. Inparticular, selective culture of CD8+, CD4+, T-cells, B-cells,macrophages and NK cells is conducted to examine the possible presenceof the CFS agent in one of these cell types. The cells are culturedselectively since one cell type may prevent replication of the agent inanother cell type. For example, removal of CD8+ cells from some PMCcultures has enabled recovery of HIV from the cells when the virus couldnot be detected otherwise (Walker, Science (1986) 234:1563 andImmunology (1989) 66:628).

The cell selection and depletion is undertaken using immunomagnetic (IM)beads (Gaudemack, et al., J. Immunological Methods (1986) 90:179). ThePMC are isolated from each sample using monoclonal antibody-coated beads(Dynabeads, M-450 Dynal, Oslo, Norway) with selectivity for the majorhuman cell subsets. Twenty to fifty million PMC are suspended in 4-5 mlof phosphate buffered saline (PBS) containing 2% heat-inactivated (30min., 56° C.) fetal calf serum (FCS). This single cell suspension isadded to the IM beads (previously washed 3 times with cold PBScontaining 2% FCS) to yield a bead:target cell ratio of approximately3:1. The bead cell suspension is incubated at 4° C. on a rotatingrocking machine for 30-35 min. Cells rosetting with IM beads arecaptured by placing the tube in a magnetic device (Dyno magneticparticle concentrator) for 1-2 min. followed by removal of thesupernatant containing the non-bound cells. The rosetted cells arewashed 3 times with cold PBS containing 2% FCS using the magneticcapture device noted above and then counted. The non-adherent cells aresubsequently treated with other IM beads coated with a differentmonoclonal antibody and the procedure repeated.

This approach with IM beads can also be performed by first reacting thePMC with mouse monoclonal antibodies to specific cell surface markers.Then, beads with antibodies to mouse immunoglobins are added to selector deplete a specific cell type. The purity of the subset is analyzed bydouble-staining procedures on a Becton-Dickinson FACscan using specificmonoclonal antibodies (Levy, et al., Clin. Immunol. Immunopathol.,(1985) 35:328).

2). All cells obtained from the blood are cultured in the presence ofIL-2 (Pharmacia, Silver Spring, Md.) except for macrophages. They aregrown in 10% FCS and 5% human (AB+) serum as described in Homsy, et al.,Science, (1989) 244:1357. The cells are examined for modulation of cellsurface markers by FACS analysis (see below) and for morphologicchanges. The cells and fluids are monitored for an infectious agentevery 3 days, for up to 28 days by the procedures listed in Appendix 6.This period of time has been found necessary for detection of HIV in thecells of some infected individuals (Castro, et al., J. Clin. Microbiol.,(1985) 26:2371). The cells are activated with PHA, pokeweed mitogen(PWM), or another mitogen specific for each cell type. Supernatants fromthe cultures are obtained and passed to fresh cells from normalcontrols.

Included in these studies is the cultivation of the different cells ortheir fluids with established human T, B, and macrophage cell lines, andanimal cell lines (see Appendix 3). Induction of CPE, RT activity, orspecific IFA staining would be used to detect infection. Finally, toaugment detection of an agent, halogenated pyrimidines (e.g., IUDR) toactivate virus replication are used.

f. Specific Studies of Macrophages

In the initial studies (see Preliminary Studies, Section C) CPE innormal macrophages inoculated with supernatant fluid from the PMCculture of an individual with CFS was found. To rule out a cytokineeffect, filtered material from these cultures was then passed to freshmacrophages and CPE was again noted as demonstrated by cell death andgranular formation in the cells. However, continued passage of thisCPE-inducing effect was not achieved.

These studies are being repeated using macrophages of CFS patients as asource of supernatant fluid. They are obtained by adherence to plasticas described in Homsy, et al., Science, (1989) 244:1357. Trypsinizationis used to maintain purity of the cells. Cells are followed for thepresence of an infectious agent as described (see Appendix 6). Inaddition, fluids from the macrophages as well as CDS+, CD4+, B cells andNK cells from CFS patients are inoculated onto macrophages from controlindividuals to evaluate whether there is a transferable morphologiceffect on these cells in culture.

The culture supernatants from CFS macrophages are also inoculated onto aselection of animal and human cells (see Appendix 3) and their possibleinduction of CPE, RT activity, and antigen is evaluated as noted above.

g. Specific Studies with NK Cells

A common finding in CFS is decreased NK cell function (Kibler, et al.,J. Clin. Immunol. (1985) 5:46; Murdoch, NZ Med J (1986) 1015511;Caligiuri, et al., J. Immunol (1987) 139 (10):3306; Klimas, et al., J.Clin. Micro. (1990) 28(6):1403). Therefore, a patient's NK cellspurified by CD16/CD56 IM beads, or the cell culture fluid is coculturedwith normal NK cells to transfer an infectious agent detected asdescribed (see Table 6). These normal NK cells are assessed for functionby measuring their ability to kill ⁵¹ CR-labelled K562 cells (Baron, etal., Diagn. Immunol. (1985) 3:197; Hudson, et al., Practical Immunology,2nd Ed. Blackwell Scientific Publications, Oxford (1980), (see SectionC)).

h. Specific Studies with CD8+ Cells

We have found that the CD11b+ cells (suppressor CD8+ cells) decreaseduring CFS. We plan to select for the CD8+/CD11b+ cells with IM beadsand examine them for a possible infectious agent. The procedures aresimilar to those described above for NK cells. In addition, supernatantfrom the CD11b+ cells is removed and added to CD11b+ cells of normaldonors to see if any cytopathic effects are noted that could explain thereduction in this particular cell subset. An effect on expression ofcell surface markers is measured by FACS analysis. Cells are monitoredfor an infectious agent as described in Table 6. In addition, an effectof the cell supernatant from CD11b+ cells on mitogen-inducedproliferation of normal lymphocytes and their production of gammainterferon is evaluated (Klimas, et al., J. Clin. Micro. (1990)28(6):1403; Hudson, et al., Practical Immunology, 2nd Ed. BlackwellScientific Publications, Oxford (1980). (see Section C)).

4. PCR Analysis

To evaluate whether an occult retrovirus might be the cause of CFS, PCRanalysis is performed on PMC specimens from CFS patients and controlswith gag and polymerase probes of HTLV-1, HTLV-II and HIV. In brief;2×10⁶ cells are extracted and the PCR performed on the DNA as describedin Ou, C--Y, et al., Science (1988) 239:295, Innis, PCR Protocols,Academic Press (1990). Two separate gag specific primer base pairs forHIV are used: SK38/39 and SK101/145. A set of gag primers, SG166/SK296,and a set of pol primers, SK110/111, for HTLV-I is used. The Cetusthermocycler is used for 30 cycles each for 30 sec at 95°, 55° and 72°C. respectively. The amplified product is hybridized to oligomer probesfor HIV-1 (SK19 and SK102) and probes to HTLV-I gag (SG242) and pol(SK112, 1SK18). The resulting products are analyzed by polyacrylamidegel electrophoresis and autoradiography.

Similar PCR evaluation is performed using DNA probes characteristic forother viral families such as parvoviruses, herpes viruses, papillomaviruses, and enteroviruses.

All publications and patent applications cited in this specification areherein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method for aiding in the diagnosis of chronicfatigue syndrome (CFS), comprising the steps of:(a) obtaining a sampleof peripheral mononuclear cells from a host; (b) contacting the samplewith an immunophenotypic panel comprising monoclonal antibodies for CD8⁺antigens, CD11b antigens, CD38 antigens and HLA-DR antigens; and (c)allowing the antibodies to bind to an antigen and classifying said hostas having CFS when said sample shows at least two of the followingconditions:(i) a reduction in the percentage of CD8⁺ CD11b+ cells; or(ii) an increase in the percentage of CD8⁺ CD38+ cells, or (iii) anincrease in the percentage of CD8⁺ HLA-DR cells as compared topercentages of cells having said markers obtained using control hostsnot having chronic fatigue syndrome.
 2. The method of claim 1, whereinbinding of antibodies to said cells is measured by detecting thepresence of a fluorochrome attached to said antibodies.
 3. The method ofclaim 1, wherein evaluating said sample for CD11b cells furthercomprises determining an increase in the number of CD11b- cells.
 4. Themethod of claim 1, wherein said peripheral mononuclear cells areobtained from a blood sample from said host.
 5. A kit for aiding in thediagnosis of chronic fatigue syndrome, comprising:an immunophenotypicpanel of antibodies which includes monoclonal antibodies for CD8+antigens, CD11b antigens, CD38 antigens and HLA-DR antigens, whereineach of the antibodies of the panel is labelled so as to be separatelyidentifiable when used concurrently.